Mouse mutants provide an increasingly important resource for identification of human disease genes and genes that are important in mammalian development. Insertional mutations in transgenic mice can be useful for isolation of mutant genes. Using a transgene insertion into the neurological mutant motor endplate disease, we cloned a novel voltage gated sodium channel alpha gene, Scn8a. We propose to isolate the craniofacial development gene that is mutated in transgenic line 9257. Abnormalities in heterozygotes carrying this mutation include circling behavior, hypoplasia of the vestibular canals, nasal bones, and turbinates, and deviated nasal septum. Homozygous mutants survive to birth. We have isolated large insert genomic clones from the mouse locus and from the human homolog on chromosome 18q11. Exon amplification and cDNA selection will be used to identify transcripts. Functional analysis of the 9257 gene will include in situ hybridization to determine the expression pattern during fetal development, and immunocytochemistry with specific antibodies to analyze subcellular location. Comparison with sequences in public databases will be carried out to detect relationships to known genes. We will evaluate the contribution of the 9257 gene to the closely linked Twirler mutation, which produces circling, vestibular hypoplasia, and obesity in heterozygotes, and cleft palate in homozygotes. We will map a new dominant circling mutant to assess potential allelism with 9257 and Twirler. We will determine the feasibility of using the 9257 transgene insertion for access to the nearby obesity and ataxia (ax) genes. We developed a polymorphic (GATA)n microsatellite marker for the human locus that will be distributed for linkage testing in human pedigrees. To investigate the relationships among genes responsible for craniofacial anomalies, we will analyze the expression of functionally related genes during fetal development in transgenic homozygotes. Candidate genes mapping to the nonrecombinant region of human and mouse chromosomes 18 will be tested. New insertional mutations will also be characterized. These projects will contribute to isolation of genes involved in craniofacial development, identification of new genetic disorders, and development of the human and mouse comparative genetic maps.